Arsenic and Slavi-1, inspite of the same level of

Arsenic is taken up by the plants and accumulates indifferent tissues leading to toxicity and contamination of food chain 47, 48.Studies suggest that As treated rice genotypes differ from each other at phenotypicand genetic level with differential expression of number of genes 19, 49. However,detailed studies to elucidate processes and molecular networks involved in As(V)response using natural variation have not been carried out. In this study, theeffect of As uptake and accumulation on Arabidopsisnatural variation was investigated in contrasting accessions. Analysis showedvariation in the As translocation potential of Col-0 and Slavi-1, inspite ofthe same level of As accumulation in the root tissue. (Fig. 2C).

This suggeststhat natural variation in As transport and accumulation mechanism might beregulating the differential response in these accessions. Genome-widetranscriptional profiling identified set of genes, which might be involved in thecontrasting response of accessions towards As(V) stress. In Col-0, severalgenes related to stress response, osmoprotectants and antioxidant system werefound to be up regulated (Supplementary Fig.

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S4). However, in Slavi-1,stress-responsive genes such as HSPs, GSTs, and CYPs were modulated. Studieshave reported modulated expression of HSPs, CYPs and GSTs in response to heavymetal stress in different plant species 18, 50, 51, 52. In this study, GSTsfrom different classes were observed to be up regulated (Supplementary Fig. S3).A number of differentiallyexpressed genes encoding transporter proteins were identified in Col-0 (SupplementaryFig.

S4) and Slavi-1 (Supplementary Table S8). Genes associated with transportof sulfate 53, metals 54, 55, sugar 56 and lipid transfer proteins areknown to play key role in heavy metal stress in plants. The modulation in expressionof sugar transmembrane transporters, AtSWEET2and AtSWEET16, in As tolerantaccession Col-0 suggest the restricted release of sugars especially glucose inresponse to As stress, which may restricts sugar efflux from the cells inresponse to As stress thereby conferring As tolerance. Further, the expressionof phosphate transporters such as PHT4;1 and PHT2;1 are down-regulated inCol-0. Studies suggest the important role of PHT4;1 in chloroplast  compartmentation of phosphate and ATPsynthesis 57. Since it plays important role in shoot, its down-regulation inCol-0 may limit root to shoot transportation of As(V), thereby minimizing thesensitivity in As- tolerant accession as compared to Slavi-1. Expression of lipidtransfer proteins is known to be induced during stress owing to rapidperoxidation due to the production of reactive oxygen species 47.

These weredown-regulated in Col-0 whereas up-regulated in Slavi-1, suggesting stressperception is higher in Slavi-1, leading to increased production of ROS. Contrary,in Slavi-1, enhanced expression of genes encoding major facilitator superfamilywas observed under As(V) stress. Studies suggest their role in ATP dependentefflux of xenobiotic compounds in the bacterial system 58 and provide resistancetowards stress. Further, sulfatetransporters were highly up-regulated in Slavi-1 as compared to Col-0. It hasbeen reported that sulfate enters via sulfate transporter and contributes insynthesis of metal chelating molecules such as GSTs and PCs for the detoxificationof toxic compounds 1, 37, 52. This enhancement is co-related with the abundanceof GST transcripts in Slavi-1, which suggests that increased GST expressionprofiles might account for As-sensitive accession Slavi-1 being highly affectedby As(V) stress.

Specificinduction of osmoprotectant-related gene, Galactinol synthase 1 (AtGolS1) in As(V)-tolerant accessionCol-0, is in accord with its role in protecting plant cells against abioticstress induced oxidative damage 59,60,61. Expression of manystress-related genes is regulated by transcription factors (TFs), viainteraction with cis-elements presentin the promoter regions. In this study, a set of regulatory genes withdifferential expression were identified in Col-0 (Supplementary Table S10) andSlavi-1 (Supplementary Fig. S3). Previous studies have identified role of somekey TFs including MYB, HSF, Zinc finger/CCCH, bHLH, Aux/IAA, WRKYs and AP2/ERFinvolved in As stress responses 33, 50, 62. In this study, expression ofmembers of MYB transcription factor family was found to be modulatedexclusively in Slavi-1. MYB genes may play important role in As(V) stresstolerance, as some of these are known to enhance anti-oxidant potential by regulatingphenylpropanoid/ flavonoids biosynthetic pathway 63,64. In addition, a numberof members of WRKY 65 and Zinc finger/C2H2 or CCCH, AP2/ERF and NAC 66, 67 weredifferentially expressed in Slavi-1.

Several transcriptomic studies on rice andArabidopsis identified the importantroles of members of these transcription factor gene families in regulatingplant responses to various stresses 51, 66. The ERF subfamily hasbeen known to participate in the ethylene signaling pathway in plants inresponse to abiotic stresses 68 including Cd and As stress in Arabidopsis and rice 33, 69. Previousstudies suggest DREB subfamily of AP2/ERF family under heavy metal stress hasinverse relation with osmotic potential in cell 70.Intriguingly, up-regulated expression of members of this gene family in Slavi-1suggests a possibility of enhanced heavy-metal inflow in the cells, which mightbe a reason for the enhanced accumulation in shoot and increased sensitivity inthis accession. Differential expressionof hormone-related genes, regulatory as well as functional components ofproteasome-related mechanisms might lead to contrasting As(V) responses ofCol-0 (Supplementary Fig. S4) and Slavi-1 (Supplementary Fig. S3).

It is reportedthat several phytohormones including ethylene play an important role during Asinduced oxidative stress 50, 71, 72. Genes encoding ethylene biosynthesis andsignaling components were significantly modulated in As-tolerant accession(Col-0) (Supplementary Fig. S4).  Studiessuggest brassinosteroids (BRs) inhibits ROS induced membrane peroxidation andenhances the production of antioxidants 73, 74. In Slavi-1, genes encodingBRs are down-regulated (Supplementary Fig. 3), which might be responsible forincreased sensitivity towards As stress. Additionally, enhanced expression ofthe components and intermediates of the ubiquitin/proteasome pathway in Slavi-1confirms extent of sensitivity in this accession. Protein modification anddegradation associated genes belonging to RING/U-box protein superfamily andRING-H2 finger protein family (Supplementary Table S4) were also up-regulatedin Slavi-1.

Taken together, our results suggest that genetic variations in accessionsleads to the induction of specific pathways along with the common networks inresponse to As(V) stress. Therefore, the studies carried out using naturalvariation 75, 76 for the genetic regulation in response to As stress andnutrient deficiency 77 opens up a new avenue for better understanding ofmechanisms of As uptake, tolerance and detoxification in plants.